1) We have launched a project to develop improved green photoactivatable fluorescent proteins (PAFPs) for Photoactivated Localization Microscopy (PALM). Currently, PAFPs are available in essentially two colors, green and red. Red PAFPs have proven to be useful for PALM by providing low backgrounds and sufficient numbers of photons capable of <25 nm uncertainty in localization. Green PAFPs suffer from low photons and high backgrounds, which limit precise molecular localization. We are currently working on developing an improved green PAFP for PALM. Current status of the project is that variants of PAGFP have been developed with low background, but at the expense of photoactivation contrast. Current efforts are aimed at reengineering by site-directed mutagenesis high photoactivation contrast while maintaining the low background fluorescence. Yan Fu is working on this project and had the assistance of Michelle Lee during the summer. Candidate molecules are currently being characterized. 2) Working with Vladislav Verkhusha (Albert Einstein College of Medicine), we have developed a farred shifted photoconvertible fluorescent protein, PSmOrange. To our knowledge this molecule has the longest excitation wavelength of the optical highlighter fluorescent proteins making it useful in in vivo animal studies. It was also found to be sufficient for PALM experiments when using our 640 nm laser excitation. This project resulted in a paper published in Nature Methods. 3) A project related to PAFPs as well as conventional fluorescent proteins, whether current or under development, is the characterization of their blinking behaviors. Ideally, PAFPs would be turned on, fluoresce for a given period of time, photobleach, and never turn on again. However, common to almost all fluorophores, PAFPs sample dark states during which they do not produce photons. This is problematic since it is difficult to determine if a molecule has blinked or photobleached, and when it turns on once again it is difficult to determine if it is the same molecule or a different molecule. We are testing variants with mutations within and around the PAFP chromophores to determine which affect the blinking on times, blinking off times, and percentage of blinking molecules in a population. This project is currently pursued by Yan Fu and Ernesto Casillas, an NIH Academy trainee. Collaborators on the project include the labs of Harald Hess (HHMI) and Jennifer Lippincott-Schwartz (NICHD). 4) A Director's Challenge Award has been awarded for a project in collaboration with Jennifer Lippincott-Schwartz (NICHD). This project,"High resolution imaging of iPS cells" is driven by Jennifer's interest in Induced Pluripotent Stem (iPS) cells as promising tools for drug development, disease modeling, and tissue repair. A new approach to super-resolution microscopy in thick specimens developed by Betzig and colleagues (HHMI, Janelia Farms, Ashburn, VA) is needed for this project. The new technology combines a very thin sheet of illumination with 2-photon excitation (2PE) and structured illumination to probe into biologically relevant 3D environments at speeds needed to visualize subcellular dynamics. Preliminary data indicate that better 2P fluorescent proteins are needed to move this project forward at a reasonable pace. Thus, fluorescent protein derivatives will be tested and further developed for optimal 2PE imaging of iPS cells. My lab's contribution will be to develop the instrumentation for screening and developing mutant fluorescent proteins for better two-photon excitation properties. This project is currently in the design phase. 5) We are collaborating with Moshe Levi (UCHSC) on a project to image with PALM the localization of sodium phosphate transporters, NaPi-2a and NaPi-2c, in the apical brush border membrane (BMM) of opossum kidney (OK) cells. Our project will require multi-color PALM experiments on NaPi molecules and their interacting partners located in the convoluted apical membrane of OK cells under several conditions affecting molecule localization, molecule diffusion, and molecule clustering. Thus, we will need to localize the molecules in 3 dimensions within >1m long membrane microvilli structures. These will be challenging experiments from both imaging and biology standpoints, but the obstacles are straightforward and we anticipate overcoming them as the project matures. Current status is developing and testing the PAFP tagged molecules expressed in COS 7 cells. This project has been transferred from Yan Fu to Kristin Hazelwood. Most of the plasmid reagents have been prepared and preliminary experiments are being performed in OK cells. Thus far, the localization of the tagged molecules is conforming to the normal localization of the NaPi proteins. 6) Our collaboration with Nihal Altan-Bonnet involves PALM imaging of plus strand RNA viral infection of cells. These viruses include picornaviruses, coronaviruses, and flaviviruses, which after infection of a cell dramatically remodel intracellular membranes into replication organelles on which RNA replication takes place. Our interest in this project is to image at super-resolution levels (<50nm) the localization of the numerous viral and host cell components on the replication organelles involved in replication at various times points post-infection. This project presents many of the same challenges as the NaPi project. This project is currently observing host cell and viral components in uninfected and Coxsackie virus infected cells. Yan Fu is now running this project. 7) We collaborate with Anamaris Colberg-Poley on super-resolution imaging of human cytomegalovirus infected cells. Our interest is gaining insight into the transfer of pUL37x1 protein from mitochondria associated membranes to the outer mitochondria membrane. These experiments are in the preliminary stages using uninfected cells expressing PAFP tagged versions of the pUL37x1. This project is currently being run by George Patterson. 8) We collaborate with Wei-Shau Hu (NCI) on a project to localize the centers of mass of several proteins and RNAs within noninfectious Human Immunodeficiency Virus (HIV) virus-like particles. For several particles developed by Wei-Shau's lab, the fluorescence labels are fusion proteins (CFP, YFP, and mCherry) target different regions particles. By two-dimensional Gaussian fitting of the fluorescence distributions, we can provide precise localization of the average localization of the molecules. This project is under the guidance of Maria Ingaramo with assistance from Mary Sun. We have also implemented fluorescence resonance energy transfer (FRET) in determining the proximity of various components. FRET is limited to <10 nm, so we will use a combination with component localization to map the particles. Future plans include performing multi-color PALM experiments using PAFP tagged versions of the particle components. 9) With Raul Rojas, a staff scientist in the lab of Lawrence Tabak, we have undertaken a project to image with PALM the localization of Golgi apparatus enzymes. These studies are intended to help in our understanding of where the enzymes are located within the Golgi and what role these locations may play in the enzymatic activity. Specifically, we are interested in whether enzymes involved in early steps in sugar modifications of proteins are located in early compartments of the Golgi and vice versa. We have performed preliminary experiments using a marker consisting of the localization domain of galactosyltransferase and the photoactivatable fluorescent protein, PAmCherry. Raul has just finished developing plasmids containing the chimeras with the full length enzymes. Preliminary experiments are scheduled to begin in 2-3 weeks.